The mechanisms of action of rabbit muscle and yeast fructose-1, 6- diphosphate aldolase have been investigated utilizing super 18O to determine the fate of the 2-keto oxygen atom of fructose 1, 6- diphosphate in the aldol cleavage reaction. A Schiff-base mechanism would demand a loss of the label from (2-super 18O)fructose 1, 6- diphosphate to the medium producing unlabeled dihydroxyacetone phosphate, while a metal-chelate mechanism should lead to isotope retention. Dihydroxyacetone phosphate produced in the aldolase reaction was converted to alpha-phosphoglycerol via the action of alpha- phosphoglycerol dehydrogenase to prevent exchange of the keto oxygen atom. Super 18O abundances were measured by combined gas chromatography-mass spectrometry of bis-(tert-butyldimethylsilyl) alpha- phosphoglycerol dimethyl ester. For rabbit muscle aldolase, no super 18O was found in the final product. However, for yeast aldolase, approximately 68% of the super 18O was retained in the product. These data are consistent with the proposed mechanisms for these enzymes, i.e., the formation of a Schiff-base intermediate for the rabbit muscle enzyme and a metal-chelate intermediate for the yeast enzyme. Experiments designed to determine the reactions which lead to loss of super 18O in the case of yeast aldolase show this loss to be primarily the result of exchange of (2-super 18O)dihydroxyacetone phosphate with water prior to reduction by alpha-phosphoglycerol dehydrogenase. Work currently underway involves similar investigations with fructose diphosphate aldolases from a variety of sources, including spinach, Lactobaccilus casei, Escherichia coli, Clostridium perfringens and Euglena gracilis. For most of these, existing physical and chemical evidence does not clearly place them in either Class I or Class II and therefore they are of particular interest. For others, i.e., E. coli and Euglena, studies by others indicate that perhaps both classes of enzymes are produced, depending upon the growth conditions. Other types of aldolases will be investigated in a similar manner in order to characterize these enzymes as to their mechanisms of action and gain further insight into the overall utility of aldolases.